Holocarboxylase synthetase (HCS) catalyzes the binding of the vitamin biotin to carboxylases and histones. Carboxylases mediate essential steps in macronutrient metabolism. For example, propionylCoA carboxylase (PCC) catalyzes the carboxylation of propionyl-CoA in the metabolism of oddchain fatty acids. HCS comprises four putative domains, i.e., the N-terminus, the biotin transfer/ATP binding domain, a putative linker domain, and the C-terminus. Both N-and C-termini are essential for biotinylation of carboxylases by HCS, but the exact functions of these two domains in enzyme catalysis are unknown. Here we tested the hypothesis that N-and C-termini play roles in substrate recognition by HCS. Yeast-two-hybrid (Y2H) assays were used to study interactions between the four domains of human HCS with p67, a PCC-based polypeptide and HCS substrate. Both N-and C-termini interacted with p67 in Y2H assays, whereas the biotin transfer/ATP-binding and the linker domains did not interact with p67. The essentiality of N-and C-termini for interactions with carboxylases was confirmed in rescue experiments with mutant Saccharomyces cerevisiae, using constructs of truncated human HCS. Finally, a computational biology approach was used to model the 3D structure of human HCS and identify amino acid residues that interact with p67. In silico predictions were consistent with observations from Y2H assays and yeast rescue experiments, and suggested docking of p67 near Arg508 and Ser515 within the central domain of HCS.

The endoplasmic reticulum (ER) in Saccharomyces cerevisiae is largely divided between perinuclear and cortical compartments. Yeast Nvj1 localizes exclusively to small patches on the perinuclear ER where it interacts with Vac8 in the vacuole membrane to form nucleus-vacuole (NV) junctions. Three regions of Nvj1 mediate the biogenesis of NV junctions. A membrane-spanning domain targets the protein to the ER. The C-terminus binds Vac8 in the vacuole membrane, which induces the clustering of both proteins into NV junctions. The luminal N-terminus is required for strict perinuclear localization. Three-dimensional cryo-electron tomography reveals that Nvj1 clamps the separation between the two nuclear membranes to half the width of bulk nuclear envelope. The N-terminus contains a hydrophobic sequence bracketed by basic residues that resembles outer mitochondrial membrane signal-anchors. The hydrophobic sequence can be scrambled or reversed without affecting function. Mutations that reduce the hydrophobicity of the core sequence or affect the distribution of basic residues cause mislocalization to the cortical ER. We conclude that the N-terminus of Nvj1 is a retention sequence that bridges the perinuclear lumen and inserts into the inner nuclear membrane.Key words: autophagy, microautophagy, nuclear envelope, nucleus, nucleus-vacoule junctions, vacuole, yeast The endoplasmic reticulum (ER) is an interconnected network of membranes that encircles the nucleus and extends to the cell cortex. The ER is topologically continuous and shares a common lumen, yet it is divided and further subdivided into increasingly smaller and functionally specialized domains (1-3). In this fashion, many of the diverse functions of the ER, such as protein and lipid biogenesis, vesicular and non-vesicular trafficking, ion homeostasis and intracellular signaling, are segregated into a mosaic of temporally and spatially dynamic ER compartments. A significant amount of current effort is focused on understanding the structure, function and biogenesis of ER subdomains.The nuclear envelope is an especially interesting ER compartment because it houses the genome and regulates many aspects of gene expression. The topology of the nuclear envelope is unusual; it consists of two concentric membrane sheets connected by wormhole-like channels called nuclear pores. The inner nuclear membrane is associated with chromatin and, in higher eukaryotes, is linked to the nuclear lamina (4). The fungal nuclear envelope associates with chromatin but lacks a classical nuclear lamina. The outer nuclear membrane is mostly rough ER and, as such, is studded with ribosomes in the process of translating membrane or secreted proteins.The ER communicates with organelles such as mitochondria and the plasma membrane through membrane contact sites (5), which may be mediated by stable protein-protein interactions (6,7). The Saccharomyces cerevisiae nuclear envelope forms unique membrane contact sites with the vacuole called nucleus-vacuole (NV) junctions. NV junctions are Velcro-l...

Saccharomyces cerevisiae linker histone Hho1p is not essential for cell viability, and very little is known about its function in vivo. We show that deletion of HHO1 (hho1⌬) suppresses the defect in transcriptional silencing caused by a mutation in the globular domain of histone H4. hho1⌬ also suppresses the reduction in HML silencing by the deletion of SIR1 that is involved in the establishment of silent chromatin at HML. We further show that hho1⌬ suppresses changes in silent chromatin structure caused by the histone H4 mutation and sir1⌬. These results suggest that HHO1 plays a negative role in transcriptionally silent chromatin. We also provide evidence that Hho1p hinders the de novo establishment of silent chromatin but does not affect the stability of preexistent silent chromatin. Unlike canonical linker histones in higher eukaryotes that have a single conserved globular domain, Hho1p possesses two globular domains. We show that the carboxyl-terminal globular domain of Hho1p is dispensable for its function, suggesting that the mode of Hho1p action is similar to that of canonical linker histones.

Transcriptional silencing in Saccharomyces cerevisiae is mediated by heterochromatin. There is a plethora of information regarding the roles of histone residues in transcriptional silencing, but exactly how histone residues contribute to heterochromatin structure is not resolved. We address this question by testing the effects of a series of histone H3 and H4 mutations involving residues in their aminoterminal tails, on the solvent-accessible and lateral surfaces of the nucleosome, and at the interface of the H3/H4 tetramer and H2A/H2B dimer on heterochromatin structure and transcriptional silencing. The general state, stability, and conformational heterogeneity of chromatin are examined with a DNA topology-based assay, and the primary chromatin structure is probed by micrococcal nuclease. We demonstrate that the histone mutations differentially affect heterochromatin. Mutations of lysine 16 of histone H4 (H4-K16) and residues in the LRS (loss of rDNA silencing) domain of nucleosome surface markedly alter heterochromatin structure, supporting the notion that H4-K16 and LRS play key roles in heterochromatin formation. Deletion of the aminoterminal tail of H3 moderately alters heterochromatin structure. Interestingly, a group of mutations in the globular domains of H3 and H4 that abrogate or greatly reduce transcriptional silencing increase the conformational heterogeneity and/or reduce the stability of heterochromatin without affecting its overall structure. Surprisingly, yet another series of mutations abolish or reduce silencing without significantly affecting the structure, stability, or conformational heterogeneity of heterochromatin. Therefore, histone residues may contribute to the structure, stability, conformational heterogeneity, or other yet-to-be-characterized features of heterochromatin important for transcriptional silencing.

Saccharomyces cerevisiae Esc2p is a member of a conserved family of proteins that contain small ubiquitin-like modifier (SUMO)-like domains. It has been implicated in transcriptional silencing and shown to interact with the silencing protein Sir2p in a two-hybrid analysis. However, little is known about how Esc2p regulates the structure of silent chromatin. We demonstrate here that ESC2 differentially regulates silent chromatin at telomeric, rDNA, and HM loci. Specifically, ESC2 is required for efficient telomeric silencing and Sir2p association with telomeric silent chromatin and for silencing and maintenance of silent chromatin structure at rDNA. On the other hand, ESC2 negatively regulates silencing at HML and HMR and destabilizes HML silent chromatin without affecting Sir2p association with chromatin. We present evidence that Esc2p is associated with both transcriptionally silent and active loci in the genome, and the abundance of Esc2p is not correlated with the chromatin state at a particular locus. Using affinity pull-down analyses, we show that Esc2p and Sir2p interact in vivo, and recombinant Esc2p and Sir2p interact directly. Moreover, we dissect Esc2p and identify a putative SUMO-binding motif that is necessary and sufficient for interacting with Sir2p and SUMO and is required for the function of Esc2p in transcriptional silencing.Transcriptional silencing in Saccharomyces cerevisiae occurs at telomeric, rDNA, HML (homothallic mating locus left), and HMR (homothallic mating locus right) loci and is mediated by a special silent chromatin akin to metazoan heterochromatin (1). Silent chromatin is associated with histone hypoacetylation, and the NAD ϩ -dependent protein deacetylase Sir2p is essential for its formation (2-4). Sir2p associates with Sir3p and Sir4p to form the Sir complex that promotes silencing at the telomeric and HM loci, and it binds Net1p and Cdc14p to form the RENT complex responsible for rDNA silencing (5-8).The establishment of silent chromatin at telomeric and HM loci is achieved via an initiation process that recruits the Sir complex to nucleation sequences, including telomeric repeats and silencers flanking the HM loci. Telomeric repeats consist of multiple Rap1p-binding sites. Rap1p, together with the Ku70-Ku80 complex that binds to chromosome ends, recruits the Sir complex to telomeres. A silencer is composed of two or three of the binding sites for origin recognition complex, Rap1p, and Abf1p. Silencer-binding factors recruit the Sir complex through a direct interaction between the origin recognition complex and Sir1p that binds to Sir4p and the binding of Rap1p to Sir3p or Sir4p. A Sir complex recruited to a silencer or telomere is believed to deacetylate histones in adjacent nucleosomes through the deacetylase activity of Sir2p (3). The deacetylated nucleosomes are thought to bind additional Sir complexes, based on the findings that the Sir complex selfinteracts and preferentially binds hypoacetylated histones. Through repeated cycles of histone deacetylation and Sir complex ...